The microbial mats are fascinating communities that are often fueled by primary CO2 fixation by cyanobacteria. These communities were dominant in the early Earth and helped to establish the oxidizing environment of the planet. We are working on microbial mats in hot springs of Yellowstone National Park, as shown in Figure 1. These mats are about 1 cm thick and the cyanobacteria are in the top ~1 mm. The mat environment is extremely interesting since the cells experience high O2 and light during the day (the mats can be 800% saturation for O2 during the day!!) and go anaerobic during the night. There is a strong metabolic switching from respiration and photosynthesis during the day to fermentation metabolism and N2 fixation during the night. Recently, we worked hard to develop molecular tools and procedures to explore the physiology of the mats with sophisticated in situ experimentation (Steunou et al., 2006; Steunou et al., 2008), and to generate axenic strains of some of the organisms (Kilian et al., 2007). In initial studies, we worked with people at TIGR to have two cyanobacterial genomes sequenced (from organisms that grow at 50-65°C) and found that the genomes appeared to be fluid (while the orthologues between the genomes were pretty similar, there was a complete lack of synteny; the two genomes were scrambled with respect to gene arrangement relative to each other) (Bhaya et al., 2007). Furthermore, we discovered that these high temperature cyanobacteria were capable of N2 fixation (Steunou et al., 2006), which was extremely surprising since others had suggested that these unicellular cyanobacteria (Synechococcus strains) that grow at the higher temperatures could not fix N2. We then measured levels of transcripts encoding the nitrogenase protein subunits (NifD) and N2 fixation in the mat over the diel cycle. While the nitrogenase transcripts and Nif proteins accumulate in the evening, as the mat becomes anoxic, the majority of the N2 fixation occurs in the morning when the mats are still anoxic, but now there is much more energy, generated from both photosynthetic electron flow and some respiration; this allows for high level N2 fixation. In other words, the cells were in a low energy state during the night (they could only do fermentation metabolism at that time), and since the nitrogenase requires a high energy input (16 ATP for the conversion of N2 to ammonium), the fixation of N2 during the evening is low. When more energy becomes available in the morning, and since the cells already have the N2 fixation machinery in place, they can fix high levels of N2. When O2 begins to accumulate in the mats as the light intensity increases, the nitrogenase activity is inhibited (the nitrogenase is O2 sensitive) and ultimately the enzyme appears to turn over (and is remade the next evening) (Steunou et al., 2008). Figure 2 depicts the level of nitrogen fixation over the diel cycle, and the conditions associated with different times of the day.

Figure 1. Alkaline Siliceous hot spring in Yellowstone National Park. These springs have microbial mats which cover the surface. The mats that we study thrive at temperatures of between 50 and 70°C.

It is also becoming clear that metabolites made and exported by one organism in the mat are used by others. In fact, it is probably most valid to consider the mat as the organism rather than the individual microbes that make up the mat. The organisms have co-evolved to generate a self-sustaining functional structure that requires the exchange of metabolites and signals in order to flourish. These 'exchange' metabolites appear to include energy sources such as H2, and fixed carbon generated by the cyanobacteria, both through photosynthetic carbon metabolism as well as fermentation metabolism. While it would take too much space to discuss the details surrounding this concept, the reader is referred to Steunou et al., 2006, 2008.

While the mats are extremely interesting from physiological and metabolic perspectives (e.g. metabolic switching over the diel cycle, metabolite exchange), they are also fascinating from the perspective of the evolution of organisms within a densely packed population of photoautotrophic, photoheterotrophic and heterotrophic microbes. The evolution of the organisms in the mats and the interactions between phage and cyanobacteria in this environment is being extensively explored by the group of Devaki Bhaya (as at the Carnegie Institution).

Figure 2. Nitrogen fixation levels over the diel cycle. The nitrogenase is not present during the day when the mats are oxic. As the mat becomes anoxic in late afternoon, the nitrogenase accumulations, but nitrogen fixation remains low. Toward the morning the sun rises, photosynthesis is initiated, energy becomes more available but the cells remain anoxic because the rate of oxygen evolution is less than the rate of oxygen consumption by respiration. In this early morning period the cells fix significant amounts of nitrogen. As the intensity of the light increases later in the morning, oxygen accumulates in the mat and the nitrogenase activity is strongly inhibited (and the enzyme appears to degrade).